When using the TaqMan® Gene Signature Plates, should I mix the reactions after adding the master mix and template to the wells containing lyophilized primers and probe?
What are the key features of a TaqMan® MGB probe?
TaqMan® MGB probes have a nonfluorescent quencher at the 3´ end and a minor groove binder at the 3´ end that increases the melting temperature (Tm) of probe, allowing the use of shorter probes. For more information on MGB probes, please consult the following manual: "Primer Express v1.5 and TaqMan® MGB Probes for Allelic Discrimination: All PCR Instruments: User Bulletin." You can find a copy on our website by entering this title in the main search box.
Answer Id: 1371
Can TaqMan® Gene Expression Assays be used in a multiplex reaction?
These assays have not been tested to run in a multiplex environment. In addition, each assay is pre-formulated with a 6-FAM™ reporter dye, so it is not possible to incorporate two different TaqMan® Gene Expression Assays into a multiplex reaction. If you would like to multiplex TaqMan® Gene Expression Assays with an endogenous control gene, our TaqMan® Endogenous Control product line offers some commonly used housekeeping genes for human, mouse, rat, and other species, and one assay that will detect 18S rRNA from all eukaryotic organisms. These assays are available with a VIC® reporter dye and in primer-limited formulations suitable for multiplexing. To see the list of these assays, please type "TaqMan Endogenous Control" in the web search form to search and it will lead you to the product page. For information about optimizing a multiplex gene expression experiment, please see "User Bulletin #5: Multiplex PCR with TaqMan® VIC® Probes (P/N 4306236B)". You can get the document by using "User Bulletin #5' as a keyword to search our web site. This User Bulletin was written for use on the ABI PRISM® 7700, but the concepts are valid for all of our real-time PCR instruments.
Answer Id: 1836
Do you offer a human beta actin probe with a reporter dye other than FAM™ dye?
We do offer two VIC® labeled beta actin probes as part of TaqMan® Endogenous Controls:
1) Human beta actin (VIC®/ TAMRA™ Probe, Primer Limited) Cat. No. 4310881E
2) Human beta actin (VIC®/ MGB Probe, Primer Limited) Cat. No. 4326315E
Please consult our Real-Time PCR Applications page for more information.
Answer Id: 1571
Can I use the TaqMan® β-actin Detection Reagents (Cat. No. 401846) as an endogenous control for my gene expression studies?
The TaqMan® β-actin Detection Reagents were designed to amplify genomic DNA, and thus are not recommended for use as an endogenous control for gene expression studies. However, we do offer other TaqMan® assay reagents that were specifically designed for that application. Two examples are: Human ACTB (Beta Actin) Endogenous Control, Cat. No. 4310881E (TAMRA™ probe) or Cat. No. 4326315E (MGB probe).
There are many factors to consider when validating a specific endogenous control. For additional information on designing gene expression assays and controls, please refer to the following documents: "Protocol: TaqMan® Human Endogenous Control Plate", and User Bulletin #2: "Relative Quantitation of Gene Expression". You can find copies of these documents on our website by entering the titles in the main Search field.
Answer Id: 1780